Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a foodgrade extractant in the food and flavor industry. Each year 10 to12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase.
Butanol may be made through chemical synthesis or by fermentation. Isobutanol is produced biologically as a by-product of yeast fermentation. It is a component of “fusel oil” that forms as a result of incomplete metabolism of amino acids by this group of fungi. Isobutanol is specifically produced from catabolism of L-valine and the yield is typically very low. Additionally, recombinant microbial production hosts, expressing a 1-butanol biosynthetic pathway (Donaldson et al., U.S. Patent Application Publication No. US20080182308A1), a 2-butanol biosynthetic pathway (Donaldson et al., U.S. Patent Publication Nos. US 20070259410A1 and US 20070292927), and an isobutanol biosynthetic pathway (Maggio-Hall et al., U.S. Patent Publication No. US 20070092957) have been described.
Biological production of butanols is believed to be limited by butanol toxicity to the host microorganism used in fermentation for butanol production. Yeast are typically sensitive to butanol in the medium. Using a screen for 1-butanol insensitive Saccharomyces cerevisiae mutants, Lorenz et al. (Molec. Biol. of the Cell (2000) 11:183-199) identified proteins that regulate polarized growth (BUD8, BEM1, BEM4, and FIG. 1), mitochondrial function (MSM1, MRP21, and HM11), and a transcriptional regulator (CHD1). They also found that 1-butanol stimulates filamentous growth in haploid cells and induces cell elongation and changes in budding pattern, leading to a pseudohyphal morphology. Ashe et al. (The EMBO Journal (2001) 20:6464-6474) found that butanol brings about a rapid inhibition of translation at the initiation step in Saccharomyces cerevisiae. The GCD1-P180 allele has a single amino acid change in Gcd1p, which is part of the eIF2B guanine nucleotide complex that is responsible for recycling eIF2-GDP to eIF2-GTP, that allows translational regulation upon butanol addition. Smirnova et al. (Molecular and Cellular Bioloty (2005) 25:9340-9340) found by using microarray analysis that with addition of fusel alcohol, there is widespread translational reprogramming in yeast. These studies all indicate the complexity of butanol sensitivity in yeast.
Van Voorst et al. (Yeast 23(5)351-359 (2006)) found that the MAP kinase of the cell wall integrity pathway, Slt2p, was phosphorylated when cells were treated with 6% ethanol. Isoamyl alcohol induces a phenotype that resembles pseudohyphae in S. cerevisiae, that requires the activity of SWE1 and SLT2 (Martinez-Anaya et al. (2003) Journal of Cell Science 116, 3423-3431, (2003); Levin, D. E. (2005) Microbiol. Mol. Biol. Rev.: 262-291). A complete set of S. cerevisiae homozygous deletions was screened for tolerance to the aliphatic alcohols ethanol, propanol and pentanol (Fuijta et al. (2006) FEMS Yeast Res 6:744-750). Mutant SLT2 deletion strains were hypersensitive to ethanol and propanol but not to pentanol,
There remains a need for yeast cells with increased tolerance to butanol, as well as methods of producing butanols using yeast host strains that are more tolerant to these chemicals. To this end applicants have Identified genes in yeast that are involved in butanol tolerance, that can be engineered to increase the level of butanol tolerance in yeast cells used for butanol production.